Regulation Of The Size And Shape Of Ferritin Nanocage

Protein cage architectures are ubiquitous and vitally important in nature.There are three unique interfaces presented by these protein cage architectures,the interior and exterior surfaces as well as the interface between subunits.The subunits that comprise the building blocks of these structures can be modified both chemically and genetically in order to impart designed functionality to different surfaces of the cage.These protein cages have demonstrated utility in modification and packing of nanoparticle,encapsulation of guest molecules,targeted delivery and release of drug due to their high symmetry,solubility,stability,and monodispersity.Although these natural assemblies can be repurposed to perform new functions,this strategy is limited to the structures of existing proteins,which may not be suited to a given application.Recently,reported approaches to designing protein cages have satisfied this requirement in different ways,such as de novo design,the use of engineered disulfide bonds,electrostatic interactions,chemical cross-links,metal-mediated interactions,or genetic fusion of multiple protein domains or fragments.There are two main challenges:heavy tasks and hard work.To overcome these limitations,it is of great importance to establish a new method for designing novel protein cage.Here,we describe an engineering strategy termed key subunit interface redesign(KSIR)that could be used for fabrication of non-native multisubunit protein architectures.The first step of KSIR calls for determination of key subunit interfaces of a target symmetric protein architecture by using crystal structural information as a guide.The second step is to identify key amino acid residues located at the key subunit interfaces.The last step corresponds to deletion or intertation residues to redesign the key subunit interface,which triggers reassembly of the protein architecture into a non-native assembly.Ferritin is a class of ubiquitous iron storage and detoxification proteins found throughout the animal,plant and microbial kingdoms.All ferritins are composed of 24 structurally identical subunits that assemble into a very robust protein cage with octahedral(432)symmetry.The external diameter of these assembled protein cages is 12 nm and the internal cavity is 8 nm.We have constructed a non-native 48-mer nanocage and a 16-mer analogue from its native 24-mer recombinant human H-chain ferritin(rHuHF)by redesigning subunit-subunit interactions.Important results are as follows:1.There are four interfaces responsible for ferritin assembly,which correspond to C2,C3,C4,and C3-C4.By comparison,we found that the C3-C4 interface has the largest total surface area among the above mentioned four interfaces in ferritin shell,suggesting that it plays an important role in controlling protein assembly.By scanning the C3-C4 interface,we identified six amino acid residues 139NEQVKA144 located at the D-helix that do not participate in subunit interface interactions.Therefore,we genetically modified ferritin subunit by individually introducing four deletion mutations(A139NE,?139NEQV,?139NEQVKA,and ?139NEQVKAIK)into a critical region of the D helix.These mutants were prepared by virtue of molecular cloning,site-specific mutagenesis,vector construction,expression,purification,characterization and crystallization.In contrast,the characteristics of A139NEQVKA are markedly distinct from that of other mutants and native ferritin,indicative of the formation of a non-native protein.Deletion of the six "silent" amino acid residues triggers the formation of a non-native 48-mer protein cage(about 17 nm)which consists of two types of non-native subunits at a ratio of 1:1.The tertiary structures of two subunits are different but with the same amino acid sequence.This deletion causes a "shift" in contacts leading to two new assemblies:an 8-mer analogue,which is favored in solution,and a 48-mer protein which exists in the crystalline state.It was observed that in solution this 48-mer protein assembly ultimately converts to the 8-mer analogue after 10 h.This engineering approach should in principle be applicable to other multi-subunit protein architectures such as Dps,heat shock protein,and the E2 protein,which would lead to the generation of a variety of non-native protein cages with different geometries.2.According to ferritin crystal structure,helix D plays a most important role in protein assembly,which is involved in interactions of two hydrophobic cores at ends of subunit.Therefore,we genetically modified ferritin subunit by introducing 16 insertion mutations(?139L,?139LN,?139LNE,?139LNEQ,?139LNEQV,?139LNEQVK,?139LNEQVKA,?139LNEQVKAI,?139LNEQVKAIK,?139LNEQVKAIKE,?139LNEQVP,?139LLLLLL,?139AAAAAA,?139PPPPPP,?138YLNEQV and ?140NEQVKA)into D helix by insertion extra amino acid residues between two hydrophobic cores in helix D.These mutants were prepared by virtue of molecular cloning and site-specific mutagenesis.Subsequently,we fabricated a novel supramolecule where 16 different subunits co-assemble into a lenticular saucer nanocage with lower symmetry.The structure of non-native nanocage is unique among all known protein cages.Inserting more than 5 amino acid residues in helix D is necessary for complete conversion of 24-mer protein assembly into its non-native 16-mer analogue,while the type of the inserted amino acid residues appears to have no effect on such conversion.This newly non-native protein can be used for encapsulation of bioactive compounds and exhibits high uptake efficiency by cancer cells.More importantly,the above strategy could be applied to other naturally occurring protein assemblies with high symmetry,leading to the generation of new proteins with unexplored functions.